HTRF assays on the new SpectraMax M5e multi-detection microplate reader

Author(s): Cathy Olsen

Publication year: 2006

Company(s): Molecular Devices Corporation

Abstract:

This Application Note from Molecular Devices describes the detection of cAMP and IP-One HTRF assays on their SpectraMax M5e multi-mode reader. Summary: HTRF assay performance on the SpectraMax M5e is demonstrated here with two different HTRF assay kits, cAMP dynamic 2 and IP-One. Gprotein coupled receptors signal through two major pathways, regulation of cAMP and increase in intracellular calcium levels mediated by IP3. IP1 is a downstream metabolite of IP3 that accumulates in signalling cells. The cAMP dynamic 2 kit includes a monoclonal anti-cAMP antibody labeled with Eu Cryptate and d2-labeled cAMP. Free cAMP produced by cells competes with cAMP-d2 for binding to the anti-cAMP Cryptate, thus an increase in cellular cAMP leads to a decrease in FRET, which is detectable as a decrease in the emission at 665 nm. The IP-One assay has a similar format and uses a monoclonal antibody specific to IP1. Standard curves for both assays are presented, and cell-based data are included for the cAMP assay. These HTRF data demonstrate the excellent dynamic range and Z'- factors2 obtainable with SpectraMax M5e.